Tyrosine phosphorylation of proteins in primary human myeloid leukemia cells stimulated by cytokines: analysis of the frequency of phosphorylation, and partial identification and semi-quantification of signaling molecules

We investigated tyrosine phosphorylation of proteins in primary human leuke mia cells stimulated by granulocyte colony-stimulating factor (G-CSF), gran ulocyte-macrophage CSF (GM-CSF), interleukin-3 (IL-3), tumor necrosis facto r (TNF), thrombopoietin (TPO) and phorbol myristate acetate (PMA) in 61 pat ients with acute myeloid leukemia (AML), nine patients with chronic myeloid leukemia (CML) in blastic crisis and four patients in chronic phase, and c ompared these data of leukemia with those of normal human immature hematopo ietic cells. These cytokines and PMA induced tyrosine phosphorylation of pr oteins in a manner characteristic for each cytokine or PMA in AML cells. G- CSF, GM-CSF and IL-3 frequently phosphorylated p92, p80, p70, p44 and p42, p95 was frequently phosphorylated by G-CSF, and was phosphorylated in one t hird of the cases by TPO. On the other hand, TNF selectively induced tyrosi ne phosphorylation of p42, and PMA selectively induced that of p44 and p42. In marked contrast to AML cells, CML cells responded poorly to cytokines w ith protein tyrosine phosphorylation, and normal human bone marrow mononucl ear cells and CD34-positive cells also showed poor response to cytokines. T he results of the immunoprecipitation studies showed tyrosine phosphorylati on of signal transducers and activators of transcription (Stat) 5 induced b y G-CSF, GM-CSF, IL-3 and/or TPO in six cases, that of extracellular signal -regulated kinase (ERK) by GM-CSF in two cases and that of p38 by TNF in th ree cases. Intracellular amount of Stat5 was markedly increased in AML cell s compared with that in CML cells and normal human bone marrow cells, where as intracellular amount of ERK and p38 was uniformly abundant in both leuke mic and normal cells. These results show cytokine-specific and amplified ty rosine phosphorylation of proteins in AML cells and suggest that amplified response might, at least in part, result from the increased amount of signa ling molecules such as Stat5. (C) 1998 Elsevier Science Ireland Ltd. All ri ghts reserved.